Iron deficiency is a serious public health problem in resource-poor areas of the world, yet little is known at the molecular level regarding the cellular adaptation to iron deficiency. We performed microarray analyses in S. cerevisiae which revealed that multiple metabolic pathways are regulated in response to iron availability. Notably, several proteins and pathways containing iron-sulfur cluster enzymes are down-regulated in yeast grown in iron-poor media. These transcriptional changes suggested that changing iron levels may alter the flux of metabolites through these pathways. Iron is an essential cofactor for enzymes involved in numerous cellular processes, yet little is known about the impact of iron deficiency on cellular metabolism or iron proteins. Previous studies have focused on changes in transcript and proteins levels in iron-deficient cells, yet these changes may not reflect changes in transport activity or flux through a metabolic pathway. We analyzed the metabolomes and transcriptomes of yeast grown in iron-rich and iron-poor media to determine which biosynthetic processes are altered when iron availability falls. Iron deficiency led to changes in glucose metabolism, amino acid biosynthesis, and lipid biosynthesis that were due to deficiencies in specific iron-dependent enzymes. Iron-sulfur proteins exhibited loss of iron cofactors, yet amino acid synthesis was maintained. Ergosterol and sphingolipid biosynthetic pathways had blocks at points where heme and diiron enzymes function, and most of these enzymes were expressed without their iron cofactors. Iron-deficient cells exhibited dramatic depletion of iron enzyme activity, but as most iron-dependent enzymes were expressed in vast excess, loss of activity during iron deficiency did not consistently disrupt metabolism. Amino acid homeostasis was robust, but iron deficiency impaired lipid synthesis, altering the properties and functions of cellular membranes.